Irena Szumiel

Application of the comet assay for monitoring DNA damage in workers exposed to chronic low-dose irradiation. I. Strand breakage.

We examined a group of people professionally at risk of exposure to low doses of ionizing radiation (altogether 49 individuals). Age, use of therapeutic drugs, work-related exposure to hazardous agents, previous exposures to diagnostic X-rays, such as patient and nuclear medical examination, were registered. For each individual, the occupational radiation burden received over the past period of 5 years was taken from the official personal records based on film dosimetry controlled every month. A matched group of controls was chosen among the administrative employees (40 individuals). The mean age of the studied population at the time of blood sampling was 49 years (range 24-69). The individuals were divided into groups according to risk of exposure and sex. The alkaline comet assay was used to measure DNA breaks and alkali-labile sites. We compared the mean tail moments, tail length and percentage of DNA in the tail. There was a significant difference between the control and hazard groups in DNA damage. Higher DNA damage was also found for men than for women in the control group. There was no relation of DNA damage to age either in control or hazard group. Additionally, analysis of distributions of tail moment values pointed to a considerable individual diversity even in the control group. Therefore, further investigations were necessary into the suitability of the comet assay as a biological dosimetry method; the results obtained so far warrant such investigations.

Monitoring and Signaling of Radiation-Induced Damage in Mammalian Cells

This paper reviews the functions of and connections between the presumed DNA damage sensors: poly(ADP-ribose) polymerase (PARP), DNA-dependent protein kinase (DNA-PK), the protein product of the ataxia telangiectasia mutated (ATM) gene, and the tumor suppressor, p53. Recognition of DNA damage is associated with the generation of alarm signals. The possible alarm signals include synthesis of poly(ADP-ribose) polymers and initiation of phosphorylation cascades by kinases complexed with the DNA damage sensors, DNA-PK and ATM; the role of other factors is discussed, among them BRCA1 and 2, IRF-1 and RB (retinoblastoma). Alarm signal molecules generated in the cytoplasm or plasma membrane are reactive oxygen species and ceramide. Some of the signal pathways are discussed. The p53 protein, which is poised in the central junction of the postirradiation signaling, as well as p53-independent signaling pathways form an intricate network that executes concerted and partly overlapping functions in the cellular response to ionizing radiation. These functions comprise activation of specific groups of genes, control of progression through the cell cycle checkpoints, inhibition of replication and transcription, induction of apoptosis, or an adaptive response; these features of the cellular response to radiation are discussed. They affect the fate of the irradiated mammalian cell as markedly as the DNA repair efficiency. This is shown in examples of the effect of inhibition of signaling on the adaptive response of human lymphocytes and on survival of tumor cells.

Response of two strains of L5178Y cells to cis-dichlorobis-(cyclopentylamine)platinum(II). I. cross-sensitivity to cis-PAD and UV light

The response to cis-dichlorobis(cyclopentylamine)platinum(II) (cis-PAD) an antitumour platinum complex, was studied in two strains of murine lymphoma L5178Y cross-sensitive to X-rays and UV light. Dose-survival relationship, DNA synthesis formation of chromatid aberrations, progression through the cell cycle, and growth and viability changes after 1 h cis-PA; treatment at 37 degree C were examined and compared with the effects of X-rays and UV light. In both strains, cis-PAD caused immediate inhibition of progression through the cell cycle, reduced rate of DNA synthesis, delayed appearance of chromatid aberrations, and delayed death; however, there is a marked difference in sensitivity to cis-PAD between L5178Y-S strain (D0 approx. 5.8 microgram/ml) and L5178Y-R strain (D0 approx. 2.5 microgram/ml). In both strains a close resemblance was found between dose-survival relationships after cis-PAD and UV light treatment, respectively.

DNA strand breakage, cytotoxicity and mutagenicity of hydrogen peroxide treatment at 4°C and 37°C in L5178Y sublines

Abstract Cells from the L5178Y murine lymphoma subline LY-R are twofold more resistant to killing by ionizing radiation than the subline LY-S. In contrast, LY-R cells are more sensitive to killing by hydrogen peroxide. Cells of the two sublines in logarithmic growth phase were treated with hydrogen peroxide in phosphate-buffered saline for 1 h at 4°C or 37°C. From the comparison of D o values it followed that at 37°C LY-R were 3.6 times more sensitive to the killing effect of H 2 O 2 than LY-S cells; at 4°C they were 11 times more sensitive. Treatment with hydrogen peroxide at 4°C gave a considerable sparing effect, which was substantially greater for the LY-S subline; for LY-S cells D o was 5.7 times lower at 37°C than at 4°C, for LY-R cells only 1.9 times. The mutation frequency (HGPRT) in LY-R cells was increased in proportion to H 2 O 2 concentration and was the same at both treatment temperatures. In contrast, mutation frequencies initially increased, then decreased with increasing H 2 O 2 concentration in LY-S cells treated at 4 or 37°C. The concentration at which the decline was initiated was higher at 4 than at 37°C. DNA damage after H 2 O 2 treatment (both temperatures, 5 min) was estimated from the ‘comet’ assay (single-cell gel electrophoresis). The initial damage, but not the residual damage, differed significantly in LY sublines. A period of slower repair (between 3 and 10 min) was found in LY-R cells.

Ionizing radiation-induced oxidative stress, epigenetic changes and genomic instability: The pivotal role of mitochondria

Abstract Purpose: To review the data concerning the role of endogenously generated reactive oxygen species (ROS) in the non-targeted ionizing radiation (IR) effects and in determination of the cell populations fate, both early after exposure and after many generations. Conclusions: The short-term as well as chronic oxidative stress responses mainly are produced due to ROS generation by the electron transport chain (ETC) of the mitochondria and by the cytoplasmic NADPH oxidases. Whether the induction of the oxidative stress and its consequences occur or are hampered in a single cell largely depends on the interaction between the nucleus and the cellular population of several hundred or thousands of mitochondria that are genetically heterogeneous. High intra-mitochondrial ROS level is damaging the mitochondrial (mt) DNA and its mutations affect the epigenetic control mechanisms of the nuclear (n) DNA, by decreasing the activity of methyltransferases and thus, causing global DNA hypomethylation. These changes are transmitted to the progeny of the irradiated cells. The chronic oxidative stress is the main cause of the late post-radiation effects, including cancer, and this makes it an important adverse effect of exposure to IR and a target for radiological protection.

Lack of adverse effect of smoking habit on DNA strand breakage and base damage, as revealed by the alkaline comet assay

In our preceding papers [M. Wojewódzka, M. Kruszewski, T. Iwanenko, A.R. Collins, I. Szumiel, Application of the comet assay for monitoring DNA damage in workers exposed to chronic low dose irradiation: I. Strand breakage, Mutat. Res., 416 (1998) 21-35; M. Kruszewski, M. Wojewódzka, T. Iwanenko, A.R. Collins, I. Szumiel, Application of the comet assay for monitoring DNA damage in workers exposed to chronic low dose irradiation: II. Base damage, Mutat. Res. , 416 (1998) 37-57.], we evaluated the DNA breakage and base damage with the use of comet assay in a group of 49 workers chronically exposed to low doses of ionizing radiation. There was a statistically significant difference in the damage levels between the hazard and control group. In this paper we describe a confounding lack of effect of the smoking habit on the DNA damage in the tested groups. The genotoxic effect of the smoking habit, as well as its modifying effect on genome damage inflicted by other agents, have been firmly established. However, no statistically significant effect of smoking was found in our study, neither in the control nor in the hazard group. This lack of effect was seen in all DNA damage determinations, both direct (DNA strand breakage and alkali-labile lesions) and enzyme-combined (base damage) and did not depend on the comet parameters, which were taken as damage indicators.

DNA damage and repair in human lymphocytes and gastric mucosa cells exposed to chromium and curcumin.

Human population can be considered as a subject of combined exposure to chemicals. Hexavalent chromium is a well-known mutagen and carcinogen. Curcumin, a popular spice and pigment, is reported to have antineoplastic properties. The single cell gel electrophoresis (Comet assay) is a sensitive technique that allows detecting double- and single-strand DNA breaks caused by a broad spectrum of mutagens. In the present work the ability of curcumin to reduce DNA damage induced by chromium in human lymphocytes and gastric mucosa (GM) cells was investigated by using the comet assay. Chromium at 500 microM evoked DNA damage measured as significant (P < 0.001), about a two-fold increase in comet tail moment of both lymphocytes and GM cells. Curcumin at 10, 25, and 50 microM also damaged DNA of both types of cells in a dose-dependent manner: the increase in the tail moment reached about twenty times of the control value (P < 0.001). The combined action of chromium at 500 microM and curcumin at 50 microM resulted in the significant (P < 0.001) increase in the comet tail moment of both types of cells. In each case, treated cells were able to recover within 60 min. Our study clearly demonstrates that curcumin does not inhibit DNA damaging action of hexavalent chromium in human lymphocytes and GM cells. Moreover, curcumin itself can damage DNA of these cells and the total effect of chromium and curcumin is additive. Further studies are needed to establish the role of interaction of curcumin with DNA in carcinogenesis.

Calcium Antagonist, TMB-8, Prevents the Induction of Adaptive Response by Hydrogen Peroxide or X-rays in Human Lymphocytes

Treatment of human lymphocytes with hydrogen peroxide (10 microM, 30 min, 37 degrees C in PBS) or with 1 cGy X-rays evoked about a 30% decrease in the frequency of micronuclei upon subsequent X-irradiation (1.5 Gy). In addition to a lower micronuclei frequency, we also found an increase in the sedimentation distance of the nucleoids, when measured 90 min (duration of the isolation procedure carried out at 4 degrees C) after the adaptive dose (hydrogen peroxide or X-rays) and preceding the challenge dose. To test whether Ca2+ is involved in the induction of the adaptive response pathway, we treated cells with the calcium chelator, EGTA. When EGTA was given at the same time as the adaptive dose, it prevented the development of the adaptive response. In addition, the calcium antagonist, TMB-8, also prevented the development of the adaptive response as it prevented the reduction of both micronuclei and increased nucleoid sedimentation. Cellular treatment with TMB-8 increased the free [Ca2+] by 40%, when given together with hydrogen peroxide. The faster sedimenting nucleoids from adapted cells were also examined by ethidium bromide titration; there was no indication of any change in supercoil density or loop size. Psi-tectorigenin, an inhibitor of phosphatidylinositol turnover, did not modify the adaptive response, indicating that inositol (1,4,5)-trisphosphate is not involved in the induction of the adaptive response, but free Ca2+ ions are.

Signalling loops and linear pathways: NF-κB activation in response to genotoxic stress

The signalling loop concept was introduced in 1991 to explain activation of membrane and cytoplasmic kinases in response to DNA damage inflicted by ionizing radiation. Damage to the chromosomal DNA was thought to provide a primary signal and a secondary signal from a nucleus to cytoplasm was assumed. This scenario was confirmed although not as originally proposed. A complex of nuclear factor-kappaB (NF-kappaB) essential modulator and ataxia telangiectasia-mutated kinase activated by genotoxic agents is sent to cytoplasm, prompting nuclear translocation of the active transcription factor NF-kappaB. In parallel, linear signalling pathways are initiated in the cytoplasm, mostly by reactive oxygen species, resulting in NF-kappaB activation and nuclear translocation. The choice of NF-kappaB activation pathway and the extent of activation of various pathways may be influenced by the relative degree of damage inflicted by genotoxic agents in the nuclear and cytoplasmic compartments. The ultimate pattern of cellular response is determined by availability, abundance and localization of the proteins participating in the signal transduction.

Effect of signal transduction inhibition in adapted lymphocytes: micronuclei frequency and DNA repair.

Irradiation of human lymphocytes (1 cGy X rays, 37 degrees C) or their treatment with 10 microM hydrogen peroxide (30 min at 37 degrees C) evoked a ca 30% decrease in the frequency of micronuclei upon subsequent X-irradiation (1.5 Gy). The response was reflected in a lower micronuclei frequency, but no change in DNA repair rate was observed as measured by the comet assay, directly after the challenge dose. Treatment of lymphocytes with staurosporine, an inhibitor of protein kinases, or with TMB-8, a calcium antagonist, carried out in parallel with the adaptive dose prevented the development of the adaptive response measured as micronuclei frequency. In lymphocytes that were staurosporine- or TMB-8-treated and irradiated under adaptive conditions showed that the rate of DNA repair was not changed. We conclude that treatment with agents that interfere with the transduction of the signal triggered by the low dose prevents the development of the adaptive response induced by X rays or hydrogen peroxide. Lower chromosome damage revealed by the cytokinesis block-micronuclei test in the adapted lymphocytes is unrelated to DNA repair rate as measured by comet assay.